This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 2000-364614, filed Nov. 30, 2000, the entire contents of which are incorporated herein by reference.
1. Field of the Invention
The present invention relates to a nucleic acid detection method and apparatus for detecting presence/absence of a nucleic acid having a particular sequence in a subject substance, and a vessel for detecting the nucleic acid.
2. Description of the Related Art
In recent years, a gene detection technique by a nucleic acid immobilized array (DNA array) has attracted attention (Beattie et al. 1993, Fodor et al. 1991, Khrapko et al. 1989, Southern et al. 1994).
For example, as shown in FIG. 20, the DNA array is constituted of a DNA array 161 of glass or silicon of several centimeters square in which 101 to 105 types of nucleic acid probes different from one another in sequence are immobilized. A subject gene labeled beforehand with fluorochrome, radioactive isotope (RI), or the like is reacted on the DNA array, or a mixture of an unlabeled subject gene and labeled oligonucleotide is reacted with sandwich hybridization. When a sequence complementary with the nucleic acid probe on the DNA array exists in the subject gene, a signal (fluorescent strength, RI strength) arising from a label is obtained in a particular site on the array. When the sequence and position of the immobilized nucleic acid probe are known beforehand, a base sequence present in the subject gene can easily be checked.
Since much information on the base sequence is obtained with a micro amount of samples, the DNA array is much expected not only as the gene detection technique but also as a sequence technique (Pease et al. 1994, Parinov et al. 1996).
Furthermore, a method of using a DNA array including an electrode formed by immobilizing the nucleic acid probe, and electrochemically controlling the hybridization reaction has also been reported (Heller et al. 1997). The nucleic acid has a minus charge in a phosphoric acid back bone. Therefore, when a plus voltage is applied to the electrode, the subject gene is concentrated on the electrode. With use of this method, the hybridization reaction, which has heretofore required several hours, ends only in several minutes.
However, a gene detection method using the DNA array is a method of using the fluorescence strength or the RI strength as an indication to detect hybrid formation of the nucleic acid probe and the subject gene on the DNA array. Therefore, an intricate and expensive pretreatment for labeling the subject gene with fluorochrome, RI, or the like beforehand is necessary. Moreover, since fluorescence or RI from a micro region has to be detected, a high-sensitivity fluorescence detection system having a high resolution is necessary. Accordingly, there is a problem that the system becomes complicated, large-sized, and expensive.
A further advanced gene detection technique based on an electrochemical method has also been reported (Hashimoto et al. 1994, Wang et al. 1998). This technique does not require the label of the subject gene. Moreover, for the detection, since the electric signal is measured, a complicated system such as fluorescence detection is unnecessary. In this technique, miniaturization of the system can be expected, and the technique can also be applied to the DNA array.
However, the known conventional DNA array is superior for the detection of varieties of genes with respect to one type of sample (subject substance). There is a problem that the array cannot necessarily be said to be suitable for treatment of varieties of samples.
At present, a method in which a micro titer plate and an enzyme immunoassay are combined is used in detecting the genes of varieties of samples such as an infectious disease. FIG. 21 is a schematic diagram. A micro titer plate 172 including a plurality of bottomed vessels 171 is used so that different samples can separately be injected, and a nucleic acid probe 173 is disposed beforehand in each vessel 171.
Subsequently, the different samples including a subject gene 174 labeled beforehand with the fluorochrome, RI, or the like are individually injected into the respective vessels 171. When the sequence complementary with the nucleic acid probe exists in the subject gene, the signal arising from the label (fluorescence strength, RI strength) is obtained in the vessel. It is thus detected whether or not the subject gene of each sample is a gene having the particular sequence.
However, this method works excellently in the detection of many kinds of samples, but it entails such a problem that it is not very much suitable for detecting many kinds of genes. Further, in this method, the intricate and expensive pretreatment for labeling the subject gene with the fluorochrome, RI, or the like is necessary. Furthermore, since it is necessary to detect the fluorescence of the micro region, the high-sensitivity fluorescence detection system having a high resolution is necessary. Accordingly, there is a problem that the system becomes complicated, large, and expensive.
In a personalized medical treatment required in future, it is desired to easily detect the gene by a simple equipment even with respect to varieties of samples, and there is a demand for means which can perform the detection.
An object of the present invention is to easily perform gene detection of many kinds of genes of many samples which has been difficult with a conventional gene detection technique with a simple equipment and at a high precision, and to precisely and efficiently perform gene examination expected in a tailor-made medical treatment, and the like.
In an embodiment of the present invention, an electrochemical method is used as a nucleic acid detection method. That is, a nucleic acid immobilized electrode constituted by immobilizing a nucleic acid probe onto a conductor, and a counter electrode are brought into contact with a subject substance. An electrochemical change between the nucleic acid immobilized electrode and the counter electrode attributed to hybridization reaction with the nucleic acid probe is detected in a nucleic acid in the subject substance, so that presence/absence of a particular nucleic acid in the subject substance is detected. Therefore, an intricate and expensive pretreatment for labeling the subject gene with fluorochrome, RI, and the like, or a high-sensitivity fluorescence detection system having a high resolution is unnecessary.
In another embodiment of the present invention, a plurality of vessels in which subject substances are contained are disposed, and a counter electrode is disposed on a bottom surface or an inside surface of the vessel. By a simple operation of injecting different subject substances into the respective vessels, inserting a nucleic acid immobilized electrode into each vessel, and bringing the electrode into contact with the subject substance, it is possible to separately detect an electrochemical change of each vessel. In this case, since the counter electrode is formed in the side surface or the bottom surface of each vessel beforehand, operability is enhanced and an apparatus constitution is simplified as compared with insertion of both the nucleic acid immobilized electrode and the counter electrode into each vessel.
Moreover, in another embodiment of the present invention, there is provided a method of electrochemically detecting presence of a nucleic acid having a particular sequence in a subject substance, comprising: preparing first and second vessels, wherein in the second vessel, a solution of a nucleic acid binding reagent is dispensed, and at least the second vessel has a counter electrode on a bottom surface or a inside surface thereof; inserting the subject substance into the first vessel; inserting a nucleic acid immobilized electrode having a conductor member and a nucleic acid probe immobilized on the conductor member, to the first vessel; making a hybridization reaction to occur in the first vessel; extracting the nucleic acid immobilized electrode from the first vessel and inserting it into the second vessel; and applying a predetermined voltage between the nucleic acid immobilized electrode and the counter electrode so as to measure an electrochemical signal of the nucleic acid binding reagent solution.